Synthetic analogs of thrombospondin and therapeutic use thereof

ABSTRACT

Compounds and compositions comprising fragments and synthetic analogs of human thrombospondin are provided together with methods for their use as thrombospondin-like agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/450,738,filed on May 25, 1995, now abandoned which is a continuation ofapplication Ser. No. 08/185,614, filed on Jan. 24, 1994, now abandoned,which is a divisional of application Ser. No. 08/024,436, filed on Mar.1, 1993, now abandoned, which is a continuation-in-part of applicationSer. No. 07/587,197, filed on Sep. 24, 1990, now U.S. Pat. No.5,190,920, issued on Mar. 2, 1993, and application Ser. No. 07/483,527,filed on Feb. 22, 1990, now U.S. Pat. No. 5,190,918, issued on Mar. 2,1993.

TECHNICAL FIELD

The present invention relates generally to peptide fragments andsynthetic analogs of thrombospondin (TSP) which retainthrombospondin-like activity. The peptides retain and mimic thebioactivity of TSP as a potent promoter or inhibitor of cell adhesionand attachment to different cell lines. The peptides find use as agentsin inhibiting metastasis since TSP has previously been shown to mediatetumor cell metastasis presumably by mechanisms involving the celladhesive domain of TSP. These peptides also find use in differentbiological and pharmaceutical applications such as: (a) promoting andinhibiting cellular attachment to tissue culture flasks, (b) promotingwound healing, angiogenesis, and implant acceptance, (c) use as agentsfor anti-platelet aggregation, (d) use as agents for antimalarialactivity, and (e) use as diagnostic reagents in different therapeuticapplications.

BACKGROUND

Thrombospondin (also known as thrombin sensitive protein or TSP) is a450,000 molecular weight protein composed of three identicaldisulfide-linked polypeptide chains (Lawler et al. J. Cell Biol (1986)101:1059-71). TSP is secreted by platelets in response to physiologicalactivators such as thrombin and collagen (Lawler, J. Blood (1986)67:112-123). TSP comprises 3% of the total platelet protein and 25% ofthe total platelet alpha granular protein (Tuszynski, G.P. et al. (1985)J. Biol. Chem. 260:12240-12245). Other cells also synthesize TSPincluding fibroblasts (Jaffe, E. A. et al., (1983) Natl. Acad. Sci. USA80:999-1002), smooth muscle cells (Raugi, G. J. et al., (1982) J. CellBiol. 95:351-354), and endothelial cells (McPhearson, J. et al. J. Biol.Chem. 256:11330-11336). TSP has been found in certain tumor tissues,such as melanoma cells (Varani, J. et al., (1989) Clin. Expl. Metastais7:319-329), squamous lung carcinoma (Riser, B. L. et al., (1988) Exp.Cell Res. 174:319-329) and breast carcinoma (Pratt, D. A. et al., (1989)Eur. J. Cancer Clin. Oncol. 25:343-350). In addition, the followingtumor cells in culture have been shown to synthesize TSP: fibrosarcoma,rhabdomyosarcoma, glioblastoma, Wilm's tumor, neuroblastoma,teratocarcinoma, choriocarcinoma, melanoma, and lung carcinoma (Mosher,D. F., (1990) Annu. Rev. Med. 41:85-97). A number of recent studies haveshown that TSP plays a major role in cell-cell and cell substatumadhesion (Tuszynski, G. P. et al., (1987) Seminars in ThrombosisHemostasis (13:361-368, Mosher, D. F., (1990) Annu. Rev. Med. 41:85-97).TSP promotes cell attachment, platelet aggregation, and lung tumorcolony formation in a murine model of experimental metastasis(Tuszynski, G. P. et al., (1987) Science 236:1570-1573, Tuszynski, G. P.et al., (1988) Blood 72:109-115). The role of TSP in adhesion is furthersupported by the observation that the extracellular matrix of mosttissues contains TSP.

TSP is composed of linear polypeptide domains that specifically interactwith various macromolecules such as plasma and matrix components. Forexample, TSP forms a complex with heparin (Yabkowitz, R. et al. (1989)J. Biol. Chem. 264:10888-10896), fibrinogen (Tuszynski, G. P. et al.(1985) J. Biol. Chem. 260:12240-12245), collagen (Mumby, S. M. et al.(1984) J. Cell Biol. 98:10888-10896, and plasminogen (Depoli. P. et al.(1989) Blood 73:976-902). The structure of TSP is conserved amongvarious animal species as indicated by the fact that the antiobodyagainst the human protein cross-reacts with TSP from mouse, rat, pig,cow, sheep, dog, and turkey (Switalska, H. I. et al., J. Lab Clin. Med.106:690-700).

Thrombospondin has been purified by a number of procedures includingexclusion chromatography (Lawler et al., J. Biol. Chem. (1978)253:8609-16), heparin affinity chromatography (Lawler et al., Thromb.Res. (1981) 22:267-269), fibrinogen affinity chromatography (Tuszynskiet al., J. Biol. Chem. (1985) 260:12240-5), barium chlorideprecipitation (Alexander et al., Biochem. J. (1984) 217:67-71) and anionexchange chromatography with HPLC (Clezarolin et al., J. Chromatog.(1984) 296:249-56).

The complete amino acid sequence of TSP has been deduced from DNA clonesprepared by various groups including Lawler et al., J. Cell. Biol.(1986) 103:1635-48; Kobayashi et al., Biochemistry (1986) 25:8418-25;Dixit et al., Proc. Ntl. Acad. Sci. (1986) 83:5449-53; and Hennessy etal., J. Cell. Biol. (1989) 108:729-36.

Cell adhesion is critical to the development and survival ofmulticellular organisms. The process of cell adhesion is complexrequiring numerous extracellular proteins such as fibron-ectin,vitronectin, collagen, laminin, and TSP and numerous families ofcellular receptors such as the integrins and cellular adhesion molecules(CAMS). These molecules are involved in the adhesion of both normal andtumor cells and have been studied quite intensively in recent years.

The amino acid sequence, Arg-Gly-Asp (RGD), was established as a cellattachment domain in fibronectin (Pierschbacher, M. D. and Ruoslahti,E., (1984) Nature (London) 309:30-32). The same or related sequenceshave been discovered in many proteins and serve as cell binding sitesfor such macromolecules as fibrinogen (Ginsberg, M.D. et al., (1985) J.Biol. Chem. 260:11891-11896). However, it appears that the adhesivefunction of laminin may not be based on the RGD sequence, but on apeptide segment of the B1 chain containing the amino acid sequencetyrosine-isoleucine-glycine-serine-arginine (YIGSR) (Sasaki, M. 1987,Proc. Natl. Acad. Sci. 84:935-938). Synthetic peptides containing theRGD and YIGSR sequence promote cell adhesion.

The therapeutic use of synthetic peptides based on the adhesive domainsof fibronectin and laminin have recently been reported. Humphries et al.(2986) Science 233:467-470) were the first to demonstrate thatco-injection of the pentapeptide GRGDS with B16-F10 murine melanomacells dramatically inhibited the formation of lung colonies in C57BL/6mice. Another synthetic peptide which was derived from laminin (YIGSR)also dramatically inhibited B16-F10 melanoma cell metastasis in C57B1/6mice (Kleinman, H. K. et al., (1987) Science 238:1132-1133; Kleinman, H.K. et al., (1990) Proc. Natl. Acad. Sci. USA 87:2279-2283). Theinhibitory activity of these peptides may be due to competition withendogenous laminin and fibronectin-dependent adhesion of tumor cells tothe vascular bed of the target organ during the metastatic disseminationof the tumor cells.

Because metastasis is a step-by-step process involving the transfer oftumor cells from one site to another through the lymphatic and bloodcirculation and platelet reduction in animals effectively blockedmetastasis in animals (Gasic et al, (1968) Proc. Natl. Acad. Sci. USA48:46-52), platelets have been thought to play a special role in thedevelopment of metastasis. Since TSP comprises 25% of the total alphagranular platelet secreted-protein, TSP would be expected to have amajor role in the hemotagenous transfer of tumor cells to distantorgans. Indeed, TSP has been shown to promote tumor cell metastasis in amurine model (Tuszynski et al, (1987) 47:4130-4133). In addition, eventswhich accompany platelet activation, such as: secretion of adhesiveproteins, platelet aggregation, activation of proteolytic enzymes, andactivation of the clotting cascade have all been shown to play asignificant role in tumor cell metastasis (Gasic, G. J., (1984) CancerMetastasis Rev. 3:99-116).

Adhesive proteins which are part of the extracellular matrix control themovement, growth, and morphology of many cell types. Extracellularmatrix proteins interact with tumor cell receptors and affect tumor celladhesion to basement membrane collagen in different ways. For example,exposure of melanoma cells in vitro to laminin resulted in increasedcapacity of tumor cells to adhere to the basement membrane and toproduce lung tumor colonies (Barsky, S. H. et al., (1984) J. Clin. Inv.74:843-848; Terranova, V. P. et al., (1984) Science 226:982-985).

In view of the information described above, TSP may play an importantrole in many diverse and clinically important processes, such as: cellmigration, wound healing, nerve regeneration, and tumor cell metastasis.To better understand the pathophysiology of these processes at themolecular level, assignment of each of the biological activities of TSPto a specific subdomain or oligopeptide of TSP would provide valuableinformation. Specifically, detailed knowledge of the structure ofdomains of the TSP and TSP receptors could be used to design TSPantagonist peptides which could block pathophysiological activities ofTSP such as TSP-dependent tumor cell metastasis formation.

TSP contains three homologous peptide sequences designated type I, II,and III repeats (Lawler and Hynes, (1986) J. Cell Biol. 103:1635-1648).The three repeats consist of approximately 60 amino acids eachcontaining six cysteine residues. Type I repeats exhibit homology withpeptide segments found in a number of diverse proteins. We haveidentified two hexapeptide sequences in TSP (CSTSCG and CSVTCG) that areeither totally conserved in other proteins or present with one or twoconservative amino acid substitutions. The prevalence of the conservedsequences [SEQ ID NO.:24] is indicated in Table I below.

                  TABLE I                                                         ______________________________________                                        Protein     Sequence  Reference                                               ______________________________________                                        TSP         CSVTCG    Lawler et al,. (1986),                                              CSTSCG    J. Cell Biol. 103:1635-48                               Circumsporozoite                                                                          CSVTCG    Dame J. B. et al., (1984)                                                     Science 225:593-599                                     Trap        CSVTCG    Robson K. J. H. et al., (1988)                                                Nature 335:79-82                                        Properdin   CSVTCG    Goundis, D. et al. (1988)                                                     Nature 335:82-85                                        Glycoprotein E                                                                            CVVTCG    McGoech D. J. et al, (1985)                             Herpes simplex I      J. Mol. Biol. 181:1-13                                  Cytochrome C                                                                              CSETCG    Lawson, J. E. et al., (1985)                            oxidase               Curr. Genet. 9:351-360                                  polypeptide II                                                                Respiratory CSVTCK    Blasco, F. et al. (1989)                                nitrate reductase     Mol. Gen. Genet. 218:249-256                                                  beta chain                                              Bird spider 18S                                                                           CSVSCG    Hendriks L. et al., (1989)                              ribosomal RNA         Eur. J. Biochem. 177:15-20                              Chicken alpha                                                                             CSVVCG    Lemischka I. R. et al. (1981)                           tubulin               J. Mol. Biol. 151:101-120                               Zebrafish   CSKTCG    Njolstad P. R. et al., (1990)                           homeobox gene         EMBO J. 9:515-524                                       E. coli gut CSVTCX    Yamada M. et al., (1987)                                operon                Biol. Chem. 262:5455-5463                               E. coli ATPase                                                                            CSVTCM    Kanazawa H. et al., (1981)                                                    BBRC:103:613-620                                        Rat liver   CSVGCG    Poncin J. E. et al., (1984)                             apolipoprotein A-I    Eur. J. Biochem. 140:493                                Tryptophan  CWVTCG    Brosius, J. et al., (1982)                              synthetase            Gene 17:223-228                                         Highlands J virus                                                                         CSVTCL    Ou J. H. et al., (1982)                                                       J. Mol. Biol. 156:719-730                               Human c-myb CSVTCK    Slamon D. J. et al., (1986)                             proto-oncogene        Science 233:347-351                                     Antistasin  CRKTCP    Ginsberg V. et al. (1990)                                           CRVHCP    J. Biol. Chem.                                                                264:12138--12140                                        Etp1OO      CVCECG    Tomley F. et al (1989)                                              CSATCG    5th International Coccisiosis                                       CSRTCG    Conference 469-573                                                  CSEQCG                                                            Human desmin                                                                              CSVTCH    Li Z. et al., (1989)                                                          Gene 78:243-254                                         Human related                                                                             CSVPCG    May F. E. B. et al., J. Virol                           mammary tumor         60:743-749                                              virus                                                                         Human proto-                                                                              CSRTCG    Ouweland A. M. W. et al.,                               oncogene c-fes/fps    EMBO J. 4:2897-2903                                     Platelet GPIIb                                                                            CSVTCR    Bray P. F. et al., (1987)                                                     J. Clin. Invest. 80:1812-1817                           ______________________________________                                    

The present invention provides thrombospondin fragments and analogswhich mimic or inhibit the biological activity of intact thrombospondinwhich find use in a variety of biological, prophylactic or therapeuticareas.

SUMMARY OF THE INVENTION

It has now been found that a class of fragments or synthetic analogsfrom a specific domain of thrombospondin have a variety of uses. Thesepeptides, by means of their adhesive activity, are capable of modifyingand inhibiting tumor cell metastasis, cell adhesion and plateletaggregation in mammals in vivo. The peptides are also useful in woundhealing, for antimalarial activity, as diagnostic reagents and in otherrelated areas. The peptides are capable of promoting cellular attachmentand therefore can be used for preparing surfaces for optimal cellculture, derivatization of various prosthetic materials, and to promotebinding of surrounding tissues. Medical devices can also be designedwhich make use of such substrates to attract cells to a surface in vivoor even to promote the growing of a desired cell type on a particularsurface prior to grafting. The TSP peptides and analogs of thisinvention have been shown to have thrombospondin-like activity.

The present invention is, therefore, in one aspect directed topolypeptide compounds having thrombospondin-like activity which areidentified by the formula [SEQ ID NO.:24]:

    R.sub.1 --Cys--X.sub.2 --X.sub.3 --X.sub.4 --Cys--R.sub.2

wherein:

R₁ is a protected or unprotected terminal amino group, includinghydrogen, amino, acetyl or at least one amino acid residue or thedesamino form thereof;

X₂, X₃, and X₄ are the same or differentneutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic orneutral/polar/small or basic/non-cyclic amino acid residues, preferablyselected from the group consisting of valine, threonine, serine, andarginine;

R₂ is a protected or unprotected terminal carboxyl group includinghydroxyl, carboxyl, or at least one amino acid residue, includingcarboxyamide or alkylamide forms thereof, preferably selected from thegroup consisting of lysine, glycine, and arginine;

the structure of the polypeptide is optionally cyclized through a bondbetween the cysteines, preferably a disulfide bond, or a bond between R₁and R₂.

Also provided in accordance with aspects of the invention arepharmaceutical compositions which contain the above-recited polypeptidecompounds together with a pharmaceutically acceptable liquid, gel orsolid carrier. Administration of therapeutically effective doses ofthese compositions can provide effective enhancement or inhibition ofthrombospondin-like activity to animals, particularly vertebrates suchas mammalian and avian hosts.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 [SEQ ID NO.:25] shows the results of HPLC analysis on peptideCSVTCG--NH₂.

FIG. 2 [SEQ ID NO.:1] shows the results of HPLC analysis on peptideCSVTCG.

FIG. 3 shows the results of HPLC analysis on peptide CSVTCG which iscyclized via a disulfide bond.

FIG. 4 shows the ability of the peptides of the invention to inhibitadhesion of melanoma cells.

FIG. 5 shows the ability of the peptides of the invention to act incollagen dependent melanoma cell adhesion.

FIG. 6 demonstrates that in vivo the peptides of the invention haveantimetastatic activity.

FIG. 7 compares the lungs of mice treated with and without the peptidesof the invention in the presence of melanoma cells.

FIG. 8 shows the ability of the peptides of the invention to inhibitADP-induced platelet aggregation.

FIG. 9 shows the ability of the peptides of the invention to inhibitcollagen-induced platelet aggregation.

FIG. 10 [SEQ ID NO.:1] shows a dose response of the ability of peptideCSVTCG to inhibit collagen-induced platelet aggregation.

FIG. 11 shows the ability of the peptides of the invention to supportthe adhesion of human platelets.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a class of fragments andanalogs of thrombospondin is provided which is capable of inhibiting ormimicing the activity of thrombospondin in mammals in vivo.

A. Definitions

"Thrombospondin-like activity" is defined herein as any activity thatmimics the known biological activities of thrombospondin. Theseactivities include cell-adhesion promoting activity, cell mitogenicactivity, cell chemotactic activities, and hemostatic activities and anyactivities that derive from these activities such as tumor cell,microbial, or parasite metastasis activity, platelet aggregatingactivity, fibrinolytic activity and immune modulation.

"Antimetastatic activity" is defined herein as the ability to prevent orgreatly reduce the extent or size of tumor cell metastasis, or inhibitor cause regression of primary solid tumors:

"Wound healing activity" is defined herein as the ability to increasethe rate at which wounds heal or to improve the results of the healingprocess (i.e., less scarring, good response to tactile stimulus, etc.)

"Angiogenesis activity" is defined herein as the ability to inhibit orenhance the formation of blood vessels or lymph vessels.

"Antimalaria activity" is defined herein as the ability to inhibiteither the cytoadherence of malarial-infected red blood cells toendothelial cells, the malarial sporozoite recognition and entry intohepatocytes, or the malarial merozoite recognition and entry into redblood cells. Antimalarial activity can be demonstrated in the form of avaccine or a therapeutic that blocks cytoadherence.

"Growth factor activity" is defined herein as the ability to inhibit orpromote cell proliferation.

"Cell adhesion activity" is defined herein as the ability to promote orinhibit the attachment of cells, preferably mammalian cells, to asubstrate.

The sequence of amino acid residues of the present polypeptidecompounds, the core pentapeptide, and preferred embodiments thereof, aredefined in terms of amino acids of certain characteristics of particularsubclasses.

Amino acid residues can be generally subclassified into four majorsubclasses as follows:

Acidic, i.e., the residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium.

Basic, i.e., the residue has a positive charge due to association with Hion at physiological pH and the residue is attracted by aqueous solutionso as to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium.

Neutral/non-polar, i.e., the residues are not charged at physiologicalpH and the residue is repelled by aqueous solution so as to seek theinner positions in the conformation of a peptide in which it iscontained when the peptide is in aqueous medium.

Neutral/polar, i.e., the residues are not charged at physiological pHand the residue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot. To fit the definition of charged, a significant percentage (atleast approximately 25%) of the individual molecules are charged atphysiological pH.

Amino acid residues can be further subclassified as cyclic ornon-cyclic, a self-explanatory classification with respect to the sidechain substituent groups of the residues, and as small or large. Theresidue is considered small if it contains a total of three carbon atomsor less. Small residues are, of course, always non-cyclic.

For the naturally occurring protein amino acids, subclassificationaccording to the foregoing scheme is as follows:

Acidic: Aspartic acid and Glutamic acid;

Basic/non-cyclic: Arginine and Lysine;

Basic/cyclic: Histidine;

Neutral/polar/small: Glycine, Serine and Cysteine;

Neutral/polar/large/non-cyclic: Threonine, Asparagine and Glutamine;

Neutral/polar/large/cyclic: Tyrosine;

Neutral/non-polar/small: Alanine;

Neutral/non-polar/large/non-cyclic: Valine, Isoleucine, Leucine andMethionine;

Neutral/non-polar/large/cyclic: Phenylalanine and Tryptophan.

The protein amino acid proline, although within the classificationneutral/non-polar/large/cyclic, is not included as an alternative due toits known effects on the secondary conformation of peptide chains.

Certain commonly encountered non-natural amino acids, such as desaminoTyrosine (des Tyr), agmatine (Agm), n-formyl tryptophan (f-Trp),alpha-aminoisobutyric acid (Aib), and safcosine (Sar), statine,ornithine (Orn), homolysine, homoserine, homoarginine, norleucine (Nle),norvaline may also be incorporated into the compounds of the invention.Desamino tyrosine is incorporated at the N-terminus. Agmatine andstatine are incorporated at the C-terminus. Based on the abovedefinition, n-formyl Trp is neutral/non-polar/large/cyclic, Sar isneutral/non-polar/small, Aib is neutral/non-polar/non-cyclic, Orn isbasic/non-cyclic, homolysine is basic/non-cyclic, homoserine isneutral/polar/small, homoarginine is basic/non-cyclic, norleucine isneutral/non-polar/large/non-cyclic, and norvaline isneutral/non-polar/large/non-cyclic.

The nomenclature used to describe polypeptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino- and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.In the amino acid structure formulae, each residue is generallyrepresented by a one-letter or three-letter designation, correspondingto the trivial name of the amino acid, in accordance with the followingschedule:

    ______________________________________                                                      Three-letter                                                                             One-letter                                           Amino Acid    Symbol     Symbol                                               ______________________________________                                        Alanine       Ala        A                                                    Arginine      Arg        R                                                    Asparagine    Asn        N                                                    Aspartic acid Asp        D                                                    Cysteine      Cys        C                                                    Glutamine     Gln        Q                                                    Glutamic acid Glu        E                                                    Glycine       Gly        G                                                    Histidine     His        H                                                    Isoleucine    Ile        I                                                    Leucine       Leu        L                                                    Lysine        Lys        K                                                    Methione      Met        M                                                    Phenylalanine Phe        F                                                    Proline       Pro        P                                                    Serine        Ser        S                                                    Threonine     Thr        T                                                    Tryptophan    Trp        W                                                    Tyrosine      Tyr        Y                                                    Valine        Val        V                                                    ______________________________________                                    

In the present application, the L-form of any amino acid residue havingan optical isomer is intended unless otherwise expressly indicated,e.g., by the symbol "[D--X_(n) ]."

Compounds within the scope of the present invention can be obtained bymodifying the disclosed formulae in numerous ways, while preserving theactivity of the polypeptide compounds thus obtained. For example, whilethe amino acids of these compounds are normally in the natural L opticalisomer form, one or more, usually two or less and preferably one aminoacid may be replaced with the optical isomer D form, or a D,L-racemicmixture can be provided in the molecules comprising the polypeptidecompound.

Additionally, a disulfide linkage may be present or absent in thecompounds of the invention, as long as activity is maintained. As oneskilled in the art would recognize, branched or cyclical chains may beproduced by the formation of a peptide bond with amino acid side groupsthat contain amino or carboxyl moieties. Amino acids containing suchside groups include, for example, glutamic acid (carboxyl group),aspartic acid (carboxyl group) and lysine (amide group). Branched orcyclical chains may also be produced through the formation of a covalentdisulfide bond between amino acid residues having sulfur-containing sidegroups, such as cysteine.

As used herein, "protected" terminal amino group, refers to a terminalamino group coupled with any of various amino-terminal protecting groupstraditionally employed in peptide synthesis. Examples of suitable groupsinclude acyl protecting groups, for example, formyl, acetyl, benzoyl,trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethaneprotecting groups, for example, benzyloxycarbonyl; and aliphaticurethane protecting groups, for example, tert-butoxycarbonyl oradamantyloxycarbonyl. Gross and Mienhofer, eds., The Peptides, vol. 3,pp. 3-88 (Academic Press, New York, 1981), disclose numerous suitableterminal amino protecting groups.

As used herein, "protected" terminal carboxyl group, refers to aterminal carboxyl group coupled with any of various carboxy-terminalprotecting groups. As will be readily apparent to one skilled in theart, suitable groups include tert-butyl, benzyl or other acceptablegroups linked to the terminal caroxyl group through an ester or etherbond.

Amino acid residues contained within the compounds, and particularly atthe carboxy- or amino- terminus, can also be modified by methylation,amidation, acetylation or substitution with other chemical groups whichcan, for example, change the circulating half-life, resistance toproteases and solubility of the compounds without adversely effectingtheir activity.

In addition to the preceding definitions, the following abbreviationshave been used throughout in describing the invention:

    ______________________________________                                        BCA        bicinchoninic acid                                                 BSA        bovine serum albumin                                               t-Boc      t-butyloxycarbonyl                                                 Bzl        benzyl                                                             °C. degrees centigrade                                                 DCM        dichloromethane                                                    DIEA       diisopropyl ethyl amine                                            DMEM       Dulbecco's minimum essential medium                                DMF        dimethyl formamide                                                 HF         hydrogen fluoride                                                  HOBT       1-hydroxybenzotriazole                                             HPLC       high performance liquid chromatography                             mBHA       methylbenzhydrylamine                                              μg      microgram                                                          μl      microliter                                                         ml         milliliter                                                         mM         millimolar                                                         nm         nanometers                                                         NMP        N-methylpyrrolidone                                                %          percent                                                            PAM        phenylacetamindomethyl                                             PBS        phosphate buffered saline                                          TFA        trifluoroacetic acid                                               ______________________________________                                    

B. Preferred Embodiments

The polypeptide compounds of the invention all contain the corepentapeptide sequence [SEQ ID NO.:24]:

    R.sub.1 --Cys--X.sub.2 --X.sub.3 --X.sub.4 --Cys--R.sub.2

wherein:

R₁ is a protected or unprotected terminal amino group, includinghydrogen, amino, acetyl or at least one amino acid residue or thedesamino form thereof;

X₂, X₃, and X₄ are the same or differentneutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic orneutral/polar/small or basic/non-cyclic amino acid residues, preferablyselected from the group consisting of valine, threonine, serine, andarginine;

R₂ is a protected or unprotected terminal carboxyl group includinghydroxyl, carboxyl, or at least one amino acid residue, includingcarboxyamide or alkylamide forms thereof, preferably selected from thegroup consisting of lysine, glycine, and arginine;

the structure of the polypeptide is optionally cyclized through a bondbetween the cysteines, preferably a disulfide bond, or a bond between R₁and R₂.

Particularly preferred are those embodiments wherein the sequence [SEQID NOS.:1, 2, 5, 18 & 26-35] is selected from the group consisting of:

    ______________________________________                                        CSVTCG                                                                        CSVTCG-NH.sub.2                                                               CSVTCG               (disulfide linked)                                       CSTSCG                                                                        CSTSCG-NH.sub.2                                                               CSTSCG               (disulfide linked)                                       CSTSCG-NH.sub.2      (blocked Cys residues)                                   CRVTCG                                                                        CRVTCG-NH.sub.2                                                               RCRVTCG              (disulfide linked)                                       CSVTCK                                                                        CSVTCR-NH.sub.2                                                               CSRTCG                                                                        CRVTCG-NH.sub.2      (disulfide linked)                                       CRTSCG-NH.sub.2                                                               CSTSCR-NH.sub.2                                                               CRVTC-NH.sub.2                                                                CSTSC                                                                         CSTSCCSVTCG [SEQ ID NO: 36]                                                   ______________________________________                                    

Compounds within the scope of the present invention can be synthesizedchemically by means well known in the art such as, e.g., solid phasepeptide synthesis. The synthesis is commenced from the carboxy-terminalend of the peptide using an alpha-amino protected amino acid.t-Butylocarbonyl (Boc) protective groups can be used for all aminogroups even though other protective groups are suitable. See Stewart etal., "Solid-Phase Peptide Synthesis," W. H. Freeman Co., San Francisco(1969) and Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963), Vale etal., Science 213, 1394-1397 (1981), and Marke et al., J. Am. Chem. Sci.103, 3178 (1981). Other preparative methods which may be employedinclude the process of Houghton Proc. Natl. ACAD Sci. 82:5132 (1981), oranother preferable synthesis procedure particularly for small branchedor cyclic chain peptides which would include conventional liquid phaseprocesses. The liquid phase process, as well as other synthesis methodsare described in "Principle of Peptide Synthesis" M. Bodansky Ed.(Spring-Verlag 1984). These and other methods of peptide synthesis arealso exemplified by U.S. Pat. Nos. 3,862,925, 3,842,067, 3,972,859 and4,105,602, 4,683,291, 4,244,946, and 4,305,872.

Conveniently, compounds may be synthesized using manual techniques orautomatically employing, for example, an Applied BioSystems 430A PeptideSynthesizer (Foster City, Calif.) or a Biosearch SAM II automaticpeptide synthesizer (Biosearch, Inc., San Rafael, Calif.), following theinstructions provided in the instruction manual supplied by themanufacturer.

Although a purity of greater than 95 percent for the synthesized peptideis preferred, lower purity may be acceptable. To obtain cyclic peptides,where for example the two cysteine amino acids are bonded or where theresidues contain a disulfide bridge which may be formed by oxidizing ofa dilute aqueous solution of the peptide with K₃ [Fe(CN)₆ ]. Other meansof cyclizing which are known in the art may also be utilized. Thestabilized cyclized peptide of the present invention can also beprepared by forming a peptide bond between non-adjacentamino acidresidue. A procedure for forming such peptide bond is provided inSchiller et al., Int. J. peptide Protein Res. (1985) 25:171.

It will be readily appreciated by those having ordinary skill in the artof peptide synthesis that the intermediates which are constructed inaccordance with the present disclosure during the course of synthesizingthe present compounds are themselves useful compounds and are thuswithin the scope of the invention.

Alternatively, selected compounds of the present invention can beproduced by expression of recombinant DNA constructs prepared inaccordance with well-known methods. Such production can be desirable toprovide large quantities or alternative embodiments of such compounds.

C. Administration

Compounds of the present invention have thrombospondin-like activity inthe intact animal. Compounds of the present invention and compositionscontaining them which are shown to have the physiological effect ofinhibiting or mimicing the effect of intact thrombospondin find use innumerous therapeutic and prophylactic applications, such as cancertherapy, malaria treatment or prevention, wound healing, thrombotic orthrombolytic conditions, angiogenesis, or cell attachment.

Thus the present invention also provides compositions containing aneffective amount of compounds of the present invention, including thenontoxic addition salts, amides and esters thereof, which may, alone,serve to provide the above-recited therapeutic benefits. Suchcompositions can also be provided together with physiologicallytolerable liquid, gel or solid diluents, adjuvants and excipients.

These compounds and compositions can be administered to animals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will range from about 1 μg to300 mg/kg, more usually 10 μg to 30 mg/kg of the host body weight.Alternatively, dosages within these ranges can be administered byconstant infusion over an extended period of time, usually exceeding 24hours, until the desired therapeutic benefits have been obtained.

Typically, such compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified. The active ingredient is often mixedwith diluents or excipients which are physiologically tolerable andcompatible with the active ingredient. Suitable diluents and excipientsare, for example, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired, the compositions maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, stabilizing or pH buffering agents, and the like.

The compositions are conventionally administered parenterally, byinjection, for example, either subcutaneously or intravenously.Additional formulations which are suitable for other modes ofadministration include suppositories, intranasal aerosols, and, in somecases, oral formulations. For suppositories, traditional binders andexcipients may include, for example, polyalkylene glycols ortriglycerides: such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%.Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders,and contain 10%-95% of active ingredient, preferably 25%-70%. These oralformulations include formulations designed to protect the peptide untilit can be absorbed.

The peptide compounds may be formulated into the compositions as neutralor salt forms. Pharmaceutically acceptable non-toxic salts include theacid addition salts (formed with the free amino groups) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups maybe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,procaine, and the like.

In addition to the compounds of the present invention which displaythrombospondin-like activity, compounds of the present invention canalso be employed as intermediates in the synthesis of such usefulcompounds.

The compounds of the invention can be homopolymerized to themselves(i.e., (peptide)n) or, heteropolymerized to one another (i.e., (peptide1-peptide 2). The compounds can also be cyclized through disulfide orother means. The compounds can also be conjugated to biocompatiblepolymeric compounds, such as BIOPOL™ (W. R. Grace & Co. Conn.).

While not wishing to be bound by any theory, it is believed that thecompositions of the invention act as agonists or antagonists to nativethrombospondin. These compounds are also believed to act as agonists orantagonists to circumsporozoite protein, thrombospondin relatedanonymous protein, antistasin, and properdin complement protein.Further, since the compounds of the invention are small in size(relative to intact thrombospondin) the properties which they exhibitare more likely to be specific in nature, as opposed to the actions ofother generally adhesive compounds such as RGD containing compounds (thesequence of which is found in over a hundred proteins) and fibronectin.The side effects of the peptide compounds of the invention are greatlyreduced when compared with these broadly adhesive compounds.

D. Use

As stated previously, the compounds of the invention can be used in avariety of biological, prophylactic or therapeutic areas. It iscontemplated that these compounds are useful in prevention or treatmentof any disease state or conditions wherein thrombospondin-like activityplays a role. These disease states and conditions include but are notlimited to, metastasis, wound healing, malaria, thrombotic conditions,angiogenesis, and cell proliferation. Antibodies directed against thecompounds of the invention are also useful as diagnostic reagents,therapeutics, or carriers of other compounds. The compounds can also beused in biomedical devices.

Numerous in vitro and in vivo assays can be used to demonstratecompounds having thrombospondin-like activity. These assays include, butare not limited to, cell adhesion assays, platelet aggregation assaysand cell proliferation assays.

METASTASIS

Metastasis is the spread of disease from one part of the body to anotherunrelated to it, as in the transfer of the cells of a malignant tumor byway of the bloodstream or lymphatics. It is believed that metastasis iseffected through a cascade mechanism which includes adhesion of tumorcells to endothelium, retraction of the endothelium, matrix degradationof the basement membrane and invasion of the tumor cells into thebloodstream. Intervention at any phase in this cascade could bebeneficial to the treatment or prevention of metastatic cancers.

The native thrombospondin molecule has been shown to potentiate tumorcell metastasis (Tuszynski et al., Cancer Research (1987) 47:4130-4133).The mechanisms by which the thrombospondin potentiation occurs are notpresently well understood.

Antimetastasis activity is characterized by the ability of the compoundsto bind to melanoma cells in vitro (Tuszynski et al., Anal. Bio. (1990)184:189-91), and the ability to reduce the size and number of tumorcolonies in vivo (Tuszynski et al. Cancer Research (1987) 47:4130-4133).

The compounds of this invention are useful as antimetastatic agents,particularly useful as antipulmonary metastatic agents. These compoundsinhibit the adhesion of metastatic tumor cells, particularly those whichare responsive to thrombospondin. The compounds also reduce tumor colonynumber as well as tumor colony size.

There are a number of mechanisms by which such antimetastatic activitycan be occurring. The peptides can be cytotoxic, or inhibit cellproliferation. As inhibitors of cell proliferation, the compounds canact to (1) inhibit mitogenesis, (2) inhibit angiogenesis, or (3)activate the complement pathway and the associated killer cells.

The compounds of the invention can also find use in biomedical devices.Since the compounds have the ability to promote the attachment ofmetastatic tumor cells, it is possible to coat a biomedical device withthe compounds to effect the removal of circulating tumor cells fromblood or lymph. The biomedical device is also useful to trap hepatomas.

Another use of the compounds is as a carrier to target toxins, drugs,hormones or imaging agents to metastatic tumor cells for diagnostic ortherapeutic purposes. These carriers would also bind to hepatomas.

WOUND HEALING

Wound healing is the closure Of wounds and can be divided into fouressential components: inflammation, angiogenesis, collagen depositionand epithelialization. All four components play a role in the healing ofwounds.

Wound healing activity is characterized by the ability of the compoundsto show angiogenic activity, the ability of the compounds to stimulatecollagen deposition and DNA synthesis in the in vivo sponge model or theability of the compounds to improve wound healing or reduce healing timein an in vivo partial or full thickness wound model.

MALARIA

Malaria is an infectious disease caused by any of various protozoans(genus Plasmodium) that are parasitic in the red blood corpuscles andare transmitted to mammals by the bite of an infected mosquito. Thecompounds of the invention can be used as antimalarial vaccines or astherapeutic agents to block cytoadherence. The peptide compounds of thepresent invention or cogeners including the peptides of the presentinvention coupled to the proper immunogenic carrier may function as apotential vaccine or treatment to prevent infection with the malarialorganism.

Antimalarial activity is characterized by the ability to inhibit eitherthe cytoadherence of malarial-infected red blood cells to endothelialcells, the malarial sporozoite recognition and entry into hepatocytes,or the malarial merozoite recognition and entry into red blood cells.

ANTI-PLATELET AGGREGATION

Platelet aggregation is a normal and beneficial process to stop bleedingof damaged tissue. However, platelet aggregation can cause problemsfollowing cardiovascular treatment such as angioplasty, thrombolytictherapy or vascular grafting. Platelets contain as much as 25% of theTSP protein in the total alpha granular platelet secreted-protein.Therefore, introduction of a peptide containing the pentapeptidesequence which is conserved in the TSP molecule and which binds toreceptors on the surface of a platelet can prevent the platelet fromaggregating and forming a clot.

A drug based on the pentapeptide is expected to be an adjunct toangioplasty and thrombolytic therapy for use with other clot-dissolvingagents which are currently in the market (e.g., tPA, streptokinan). Suchan agent does not aggravate bleeding or have the risk of side effectscommon to synthetic anti-platelet drugs. Additionally, such a peptidecan help to keep open small diameter vascular grafts (such as those usedin heart bypass surgery). Similar applications are envisioned forpatients at risk for stroke.

Anti-platelet aggregation activity is characterized by a number ofassays, including (1) inhibition of ADP or thrombin-induced plateletaggregation in washed platelets; (2) inhibition of ADP-induced plateletaggregation in platelet-rich plasma; and (3) inhibition of collageninduced platelet aggregation measured in vivo.

ANGIOGENESIS

Angiogenesis is the formation of blood and lymph vessels. Angiogenesisis essential during development, in wound healing and for the growth ofsolid tumor. Angiogenesis is a complex process, requiring the sproutingand migration of endothelial cells, their proliferation and theirdifferentiation into a tube like structure and the production of abasement membrane matrix around the vessel (Herbert et al. 1988, L.Cell, Biol. 106, 1365-1373). Angiogenesis is also essential to tumordevelopment and growth. Prevention of angiogenesis can inhibit solidtumor growth. Use of the compounds of this invention can inhibit one ormore steps in the cascade process of angiogenesis and therefore suchpeptide may be useful clinically to inhibit metastasis. The compounds ofthis invention are useful in the modulation of angiogenesis,particularly in enhancing wound healing, inhibiting or preventing tumorgrowth. Standard angiogenesis assays are well known in the art.

ADHESION AND CELL ATTACHMENT

The peptides of the present invention can be used for preparing asurface for optimal cell culture, and for prosthetic materials topromote bonding with surrounding tissue. These peptides can be useful asa cell attachment protein to provide a substrate to which cells willattach by treating a hydrophobic surface such as untreated syntheticplastic resin and especially materials which are used for differentmembrane applications, e.g., nitrocellulose or polysulfone or comparablematerial with the peptide. The cell attachment properties of thepeptides can also be used to couple polypeptides covalently to a solidsupport such as gels or synthetic resins or long chain polysaccharide.This latter approach can be used for different affinity chromatographyapplications. Another important application of using such peptides arethe use of the peptide in commercial cell attachment surfaces, whereinthe particles are coated with gelatin, making it possible to grow thesame adherent cells in a much smaller volume of media than would bepossible in dishes. Medical devices can be designed for use with suchpeptides to attach cells to the surface in vivo, or even to promote thegrowth of a desired cell type on particular surfaces prior to grafting.An example of this is attachment of islet cells to a membrane or growthof endothelial cells on a prosthetic blood vessel or vascular graft.Such peptides can find uses in coating a patch graft or the like foraiding in wound healing.

ANTIBODIES

Antibodies, both monoclonal and polyclonal, directed to peptidecompounds of the present invention are useful in isolation andidentification of the subject protein from where the peptides arederived, and the present invention also pertains to such antibodies. Toprepare antibodies, any one of a number of techniques which are known inthe art can be employed. In one such technique, polyclonal antibodiesmay be synthesized by injecting an animal (for example, a rabbit) withone or more compounds of the invention. After injection, the animalnaturally produces antibodies to these compounds. When the antibodylevel rises to a sufficient level, antibody-containing blood, calledantiserum, is then drawn from the animal, and the compound-specificantibody is isolated from other antibodies in the antiserum by any oneof a number of separation techniques (for example, affinitychomatography). Monoclonal antibodies may be prepared using thetechnique of Kohler and Milstein, Nature 256, pp. 495-497 (1975).

Compounds of the present invention can also be used for preparingantisera for use in immunoassays employing labelled reagents, usuallyantibodies. Conveniently, the polypeptides can be conjugated to anantigen by means of dialdehydes, particularly from 4 to 6 carbon atomsand aliphatic, or carbodimide. These compounds and immunologic reagentsmay be labelled with a variety of labels such as chromophores,fluorophores such as, e.g., fluorescein or rhodamine, radioisotopes suchas ¹²⁵ 1, ³⁵ S, ¹⁴ C, or ³ H, or magnetized particles, by means wellknown in the art.

These labelled compounds and reagents, or labelled reagents capable ofrecognizing and specifically binding to them, can find use as, e.g.,diagnostic reagents. Samples derived from biological specimens can beassayed for the presence or amount of substances having a commonantigenic determinant with compounds of the present invention.

Thrombospondin levels are elevated in the serum of patients withmetastatic breast and colon cancer (Tuszynski et al., ThrombosisHaemostas (1989) 62:418 and Smith et al., Proceedings AmericanAssociation of Clinical Oncology (1990) 9:6). Antibodies against thepeptides of the invention can be useful as reagents indiagnostic/prognostic assays for various types of cancer, including butnot limited to, gastrointestinal tract (gastric, colonic, and rectal)carcinomas, breast carcinomas and hepatic carcinomas.

The polyclonal and monoclonal antibodies can find therapeutic use in anumber of cancer therapies. First, the antibodies can be used tosequester thrombospondin. This is useful since thrombospondin mediatestumor cell metastasis. Second, the antibodies can be used to blockthrombospondin present on the tumor cell surface. Third, cytotoxicdrugs, hormones, or imaging agents can be coupled to the antibodies foruse in cancer therapy. Fourth, a biomedical device can be coated withthe antibodies to remove excess thrombospondin from serum or to removecells which bear thrombospondin on the cell surface.

The peptides of the invention can also be used to isolate thrombospondincell surface receptors from extracts of cells or cell membranes.Standard procedures such as affinity chromatography can be employed. Thethrombospondin cell surface receptors can be used to develop betterthrombospondin analogs or to remove excess thrombospondin from serum.

The following examples are provided by way of illustration, rather thanimplying any limitation of the subject matter.

EXAMPLES

The peptides of this invention can be synthesized by conventionalmethods of peptide synthesis. The preferred conventional methods use theprocedures described in Int. J. Pept. Proc. Res. 21, 57-63 (1983). Alsopreferred is the solid phase synthesis of Merrified, J. Amer. Chem. Soc.85, 2149-2154 (1963); Science 150, 178-185 (1965); Ibid. 232, 341-347(1986). Solid phase synthesis is generally initiated from the C-terminalof the peptide by coupling a protected alpha amino acid to a suitableresin, e.g., phenylacetamidomethyl (PAM) polystyrene resin, orp-methylbenzhydrylamine (mBHA) resin when synthesizing a peptide with aC-terminal carboxyamide. During synthesis, suitable amino acidside-chain protecting groups are used as needed. Thus, aspartic acid isprotected on the beta-carboxyl group as the benzyl ester and arginine isprotected on the guanidino group by tosyl. After the desired peptide hasbeen synthesized, the peptide is cleaved from the resin and protectinggroups are removed by treatment with a reagent such as hydrogen fluoride(HF). The peptide can then be purified by high performance liquidchromatography (HPLC) or other such methods of peptide purification.Background information on the established procedures for solid phasepeptide synthesis can be found in "Solid Phase Peptide Synthesis" byStewart and Young, W. H. Freeman & Co., San Francisco, 1969.

In accordance with the above description, the following procedures wereused for the chemical synthesis of novel synthetic peptides:

Example 1 Synthesis of the Peptide Sequence CSVTCG with C-Terminal Amide

An appropriate resin 4-methylbenzhydrylamine (MBHA) for C-terminal amidewas sealed into polypropylene mesh packets (64μ). All packets wereplaced into a common vessel with CH₂ Cl₂ and vigorously shaken to washand swell the resin. All subsequent steps involved vigorous shaking toensure adequate solvent transfer. The N-α-butoxycarbonyl was thenremoved by acidolysis using 55% trifluoroacetic acid (TFA)/CH₂ Cl₂ for30 minutes leaving the α-amino acid group in the TFA salt form. Thepackets were then washed with CH₂ Cl₂ (2x), IPA (2x), and CH₂ Cl₂ (2x)to remove excess TFA and prepare for neutralization. The IFA salt wasneutralized by washing the packets three times with 5%diisopropylethylamine in CH₂ Cl₂ for 2 minutes each. This was followedby two washes with CH₂ Cl₂ to remove excess base. Packets were thenremoved from the common vessel and added to their respective 0.2M aminoacid solutions which were prepared from computer generated informationprior to neutralization. An equal volume of 0.2M dipropylcarbodiimidewas then added to activate the coupling reaction. After coupling at roomtemperature for 1 hour, the packets were washed with dimethyl-formamideand CH₂ Cl₂ and returned to the common vessel. This process was repeatedfor each amino acid. Cleavage occurred by reacting the peptide with92.5% hydrogen fluoride/7.5% anisole at -10° C. to 0° C. over 90minutes. Anisole was used as a scavenger to react with carbocationsproduced as a result of the side chain protecting group removal. Thissolution was then removed using a strong flow of N₂ followed by the useof aspirator vacuum, while maintaining the temperature at 0° C. Residualanisole was removed with 2 ethyl ether washes. The peptide was thenextracted using 10% acetic acid.

The purity of the crude peptide was checked by analytical RP-HPLC usinga Beckman System Gold with a Vydac C-18 column at a flow rate of 1ml/min. The solvent system used was 0.05% aqueous TFA(A) and 0.05% TFAin acetonitrile (B) with a gradient of 5-65% B in 30 minutes measuringthe absorbance at 215 μm). Purification was performed on Waters deltaprep. 3,000 preparative HPLC with a Waters prep. Pak Nodule RadialCompression C18 column (25 cm×5 cm, 10-20μ). The solvent system was0.05% aqueous TFA (A) and 0.05% TFA in acetonitrile (B). Various lineargradients were used measuring the absorbance at 230 nm and collecting 40ml fraction. The fractions were then analyzed on the Beckman analyticalsystem. The desired fractions were pooled and lyophilized. The final dryproduct was analyzed one more time using analytical RP-HPLC. TypicalHPLC chromatograms for this peptide after purification are shown in FIG.1.

Example 2 Chemical Synthesis of the Peptide Sequence CSVTCG Acid

An appropriate resin phenylacetamidomethyl (PAM) for C-terminal acid wassealed into polypropylene mesh packets (64μ). All packets were placedinto a common vessel with CH₂ Cl₂ and vigorously shaken to wash andswell the resin. All subsequent steps involved vigorous shaking toensure adequate solvent transfer. The N-α-butoxycarbonyl is then removedby acidolysis using 55% trifluoroacetic acid (TFA)/CH₂ Cl₂ for 30minutes leaving the α-amino acid group in the TFA salt form. The packetswere then washed with CH₂ Cl₂ (2x), IPA (2x), and CH₂ Cl₂ (2x) to removeexcess TFA and prepare for neutralization. The TFA salt was neutralizedby washing the packets three times with 5% diisopropylethylamine in CH₂Cl₂ for 2 minutes each. This was followed by two washes with CH₂ Cl₂ toremove excess base. Packets were then removed from the common vessel andadded to their respective 0.2M amino acid solutions which were preparedfrom computer generated information prior to neutralization. An equalvolume of 0.2M diisopropylcarbodiimide was then added to activate thecoupling reaction. After coupling at room temperature for 1 hour, thepackets were washed with dimethylformamide and CH₂ Cl₂ and returned tothe common vessel. This process was repeated for each amino acid.Cleavage occurred by reacting the peptide with 91.5% hydrogenfluoride/7.5% anisole at -10° C. to 0° C. over 90 minutes. Anisole wasused as a scavenger to react with carbocations produced as a result ofthe side chain protecting group removal. This solution was then removedusing a strong flow of N₂ followed by the use of an aspirator vacuum,while maintaining the temperature at 0° C. Residual anisole is removedwith two ethyl ether washes. The peptide is then extracted using 10%acetic acid.

The purity of the crude peptide was checked by analytical RP-HPLC usinga Beckman System Gold with a Vydac C-18 column at a flow rate of 1ml/min. The solvent system used was 0.05% aqueous TFA(A) and 0.05% TFAin acetonitrile (B) with a gradient of 5-65% B in 30 minutes measuringthe absorbance at 215 nm. Purification was performed on Waters deltaprep. 3,000 preparative HPLC with a Waters prep. Pak Nodule RadialCompression C18 column (25 cm×5 cm, 10-20μ). The solvent system was0.05% aqueous TFA (A) and 0.05% TFA in acetonitrile (B). Various lineargradients were used measuring the absorbance of 230 nm and collecting 40ml fraction. The fractions were then analyzed on the Beckman analyticalsystem. The desired fractions were pooled and lyophilized. The final dryproduct was analyzed one more time using analytical RP-HPLC. TypicalHPLC chromatogram for this peptide after purification are shown in FIG.2.

Example 3 Synthesis of cyclic CSVTCG-Acid

Cyclization of CSVTCG peptide was accomplished by dissolving the crudepeptide of Example 2 (52 mg) in 500 ml deoxygenated water and the pH wasadjusted to 8.5 with 28% NH₄ OH (solution A). K₃ Fe(CN)₆ (1.75 g) wasdissolved in 100 ml deoxygenated water and the pH was adjusted to 8.5with 28% NH₄ OH. This solution is called solution B.

Solution A was dropped into solution B in 2 hours and the mixture wasallowed to stir 1 more hour. The pH was then adjusted to 4 with 10% AcOHand the solution was injected onto a prep.-HPLC. After purification a 28mg peptide of 95% purity has been recovered. The composition of thecyclic material was confirmed by analytical reverse phase HPLC and byFeb-MS. Typical HPLC chromatography is presented in FIG. 3.

Example 4 Chemical Synthesis of Additional Peptides

Following the procedures outlined in Examples 1-3 and in Int. J. Pept.Proc. Res. 21, 57-65 (1983) with appropriate modification, the followingpeptides [SEQ ID NOS.:2, 5, 18 & 26-35] were synthesized. All peptidesof the invention were tested for endotoxins using standard LAL assayprocedures and found to be endotoxin free.

    ______________________________________                                        CSTSCG                                                                        CSTSCG-NH.sub.2                                                               CSTSCG            (disulfide linked)                                          CSTSCG-NH.sub.2   (blocked Cys residues)                                      CRVTCG                                                                        CRVTCG            (disulfide linked)                                          CRVTCG-NH.sub.2                                                               RCRVTCG           (disulfide linked)                                          CSVTCK                                                                        CSVTCR-NH.sub.2                                                               CSRTCG                                                                        CRVTCG-NH.sub.2   (disulfide linked)                                          CRTSCG-NH.sub.2                                                               CSTSCR-NH.sub.2                                                               CRVTC-NH.sub.2                                                                CSTCS                                                                         ______________________________________                                    

Example 5 Adhesion of B₁₆ F₁₀ Melanoma Cells to TSP and Peptides

In this example a series of peptides were tested to determine theabilities of the peptides to bind B₁₆ F₁₀ melanoma cells as compared tothrombospondin and fibronectin. It is believed that thrombospondin actsin metastasis through its adhesive properties. An assay was developed,generally in accordance with the disclosure of Tuszynski et al. (Anal.Bio. (1990) 184:189-91) which evaluates the ability of melanoma cells toadhere to the thrombospondin fragments or analogs of the invention. Inthis assay, thrombospondin (purified by the method of Tuszynski et al.,J. Biol. Chem. (1985) 260:12240-5) and fibronectin (Sigma Chemical Co.,Missouri) served as positive controls, bovine serum albumin (BSA) (SigmaChemical Co.) served as the negative control. Thrombospondin analogs ofthe invention were synthesized as described in Examples 1-4. Twomicrograms of peptide or control proteins were air dried overnight onthe wells of a 96-well microtiter plate. Wells were then washed withHEPES-buffered saline and blocked for 1 hour with 1% fatty acid free BSAin HEPES-buffered saline.

The mouse B₁₆ F₁₀ melanoma cells were grown and harvested during logphase of growth using standard procedures. The harvested cells werewashed two times in serum-free Dulbecco's minimum essential medium(DMEM) (Flow Laboratories) and suspended in HEPES-buffered saline,containing 5 mM glucose and 100 μM MgCl₂ at a final concentration of4×10⁵ cells/ml. Of the cell suspension 200,000 cells per well was addedto each well of the microtiter dish containing the various ligands andthe dish incubated at 37° C. in a CO₂ incubator for 30 minutes.Nonadherent cells were removed by aspiration and the wells washed threetimes with 200 μl of PBS. The total cell-associated protein wasdetermined by dissolving the attached cells directly in the microtiterwells with 200 μl of the Pierce BCA working solution (Pierce Chem. Co.Booklet No. 23225 (1987)). The plate was covered with an adhesive mylarsheet (Flow Labs) and incubated at 60° C. for 30 minutes. Plates wereallowed to cool to room temperature, cover sheets were removed, and theabsorbance of each well was determined at 562 nm with a microtiter platereader (Biotek, Burlington, Vt.) Absorbances were converted to number ofadherent cells by means of an empirically determined conversion factor.

The results shown in FIG. 4 indicate that the peptides of the inventiondisplay adhesive properties.

Example 6 The Effect of Peptides on Collagen Dependent Adhesion of B₁₀F₁₀ Melanoma Cells

Two micrograms of collagen in 5 mM acetic acid were adsorbed to wellsovernight at 4° C. Wells were then washed with HEPES-buffered saline andblocked for 1 hour with 1% fatty acid free bovine serum albumin (BSA) inHEPES buffered saline. A suspension of B₁₆ F₁₀ cells in HEPES-bufferedsaline, containing 5 mM glucose (200,000 cells per well) and 100 μMMnCl₂ were preincubated for 15 minutes at 37° C. in either buffer or inbuffer containing 100 μg/ml peptide or 100 μg/ml BSA. The cellsuspensions were then added to collagen-coated wells and incubated for30 minutes at room temperature. Non-adherent cells were removed byaspiration, and adherent cells determined by measurement ofcell-associated protein as previously described in Example 5. Theresults shown in FIG. 5 indicate that the peptides of the inventioninhibit the binding of the melanoma cell to collagen.

Example 7 The Effect of Peptides on B₁₆ F₁₀ Lung Tumor Cell Metastasis

The in vivo model used to demonstrate the antimetastatic activity of thepeptide compounds of the invention is described by Tuszynski et al.(Cancer Res. (1987) 47:4130-4133). Briefly, C57 black mice wereintravenously injected with 1×10⁵ B₁₆ F₁₀ mouse melanoma cells in thepresence of either control buffer (Hepes buffered saline, pH 7.4), or 1mg of the designated peptide compound of the invention. Five or sixanimals were used for each compound. Peptides tested in the assay had noeffect on cell viability as measured by Trypan blue dye exclusion. Inaddition, the peptides at 1 mg/ml did not effect cell growth after 24hours of co-culture. After 14 days, the mice were sacrificed and thenumber of lung tumors counted.

The results shown in FIG. 6 indicate the peptides of the invention haveantimetastatic activity. The bar graphs show the mean number of lungtumors observed in the treatment groups and the error bars represent thestandard error of the mean. FIG. 7 shows representative lungs from eachof the treatment groups.

Example 8 Platelet Aggregation Assay

Platelet aggregation was monitored on a single-channel aggregometerequipped to measure luminescence (Chromo-Log, Havertown, Pa.).Platelet-rich-plasma (PRP) was obtained from whole blood anticoagulatedwith 0.38% sodium citrate by centrifugation at 150 X g for 20 minutes.

The effect of peptides on ADP-induced platelet aggregation is shown inFIG. 8. 0.5 ml aliquots of platelet-rich-plasma were aggregated bystirring with 2 uM ADP in the presence of various concentrations ofCSVTCG and GRGDS. Aggregation was measured at 37° as decrease in opticalabsorbance vs. time in a chrono-log aggregometer and is shown in FIG. 8.Peptides designated in each panel are represented by their amino acidsequence using the one letter code.

Example 9 The Effect of Peptides on Colladen-Induced PlateletAggregation

Washed human platelets were suspended in HEPES-buffered saline,containing 5 mM glucose and 350 μg/ml BSA. 0.5 ml aliquots of plateletswere aggregated by stirring with 5 μg/ml collagen in the presence of 500μg/ml of various peptides. Aggregation was measured at 37° C. asdecrease in optical absorbance vs. time in a Chrono-log aggregometer andis shown in FIG. 9. 100% aggregation was defined as the maximal decreasein absorbance measured in the absence of peptide. % of control wascalculated as the decrease in absorbance measured in the presence ofpeptide divided by the decrease in absorbance measured in the absence ofpeptide times 100. Peptides are designated under each bar graph by theiramino acid sequence represented using the one letter code.

Example 10 Dose-Response of CSVTCG on Collagen-Induced PlateletAggregation

Washed human platelets were suspended in HEPES-buffered saline,containing 5 mM glucose and 350 μg/ml BSA. 0.5 ml aliquots of plateletswere aggregated by stirring with 5 μg/ml collagen in the presence ofvarious concentrations of CSVTCG. Aggregation was measured at 37° C. asdecrease in optical absorbance vs. time in a chrono-log aggregometer andis shown in FIG. 10. 100% aggregation was defined as the maximaldecrease in absorbance measured in the absence of peptideo % of controlwas calculated as the decrease in absorbance measured in the presence ofpeptide divided by the decrease in absorbance measured in the absence ofpeptide times 100.

Example 11 Adhesion of Human Platelets to TSP and Peptides

The number of adherent platelets was essentially determined aspreviously described (Tuszynski et al., Anal. Bio. supra). Briefly, 100μl of 5×10⁵ platelets/ml washed as previously described (Tuszynski etal., (1988) Blood 72, 109-225) were added to microtiter plates, thewells of which were treated with 2 μg of peptide or protein solution(HEPES buffered saline, pH 7.4). Solutions were dried at roomtemperature after incubation in a fume hood overnight. Wells were thenwashed with HEPES-buffered saline and blocked for 1 hour with 1% fattyacid free bovine serum albumin (BSA) in HEPES buffered saline. Platelets(100 μl) were incubated in the wells for 30 minutes and non-adherentplatelets were removed by aspiration. Wells were washed 3 X with 200 μlof Hepes buffered saline, pH 7.4. The number of adherent platelets wasdetermined by measuring the platelet-derived protein using the BCAprotein assay. The results are shown in FIG. 11. The label under eachbar designates the protein or peptide used to coat the well. Proteinsused were thrombospondin (TSP), fibronectin (FN), and bovine serumalbumin (BSA). Peptides are designated under each bar graph by theiramino acid sequence represented using the one letter code. The peptidehaving blocked cys residues is given as C(x)SVTC(x)G--NH--2, where xrepresents the blocking group ACM. A suspension of platelets inHepes-buffered saline, containing 5 mM glucose and 350 μg/ml BSA (5×10⁷platelets per well) were incubated in each well for 30 minutes,nonadherent platelets removed by aspiration, and adherent cellsdetermined by measurement of cell associated protein as previouslydescribed (Tuszynski et al., (1990) 184:189-191). The data is arepresentative of 2-3 experiments and data points in each experiment arethe mean of three determinations, and the error bars represent thestandard error of the mean (SEM).

Example 12 Activity of Pentapeptides

The pentapeptides CRVTC--NH2 and CSTSC will be synthesized and testedfor activity in the assays described in Examples 5-11. The results ofthe assays are expected to support the present invention.

Example 13 Antimalarial Activity of Peptides

The peptides synthesized in Examples 1-4 will be tested for antimalarialactivity. The results of the assays are expected to show that thepeptides of the present invention exhibit inhibition of malarialactivity.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 36                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CysSerValThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CysSerThrSerCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CysValValThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CysSerGluThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CysSerValThrCysLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CysSerValSerCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CysSerValValCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CysSerLysThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysSerValThrCysXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CysSerValThrCysMet                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CysSerValGlyCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CysTrpValThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CysSerValThrCysLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CysArgLysThrCysPro                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CysArgValHisCysPro                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CysValCysGluCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CysSerAlaThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CysSerArgThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CysSerGluGlnCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CysSerValThrCysHis                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CysSerValProCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CysSerArgThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CysSerValThrCysArg                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 1 is Xaa wherein Xaa=R1."                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino Acid 3 is Xaa wherein Xaa=X2."                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 4 is Xaa wherein Xaa=X3."                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 5 is Xaa wherein Xaa=X4."                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=R2."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      XaaCysXaaXaaXaaCysXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=NH2."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CysSerValThrCysGlyXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=NH2."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CysSerThrSerCysGlyXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CysArgValThrCysGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=NH2."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CysArgValThrCysGlyXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ArgCysArgValThrCysGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=NH2."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CysSerValThrCysArgXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=NH2."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CysArgValThrCysGlyXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=NH2."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CysArgThrSerCysGlyXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 7 is Xaa wherein Xaa=NH2."                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CysSerThrSerCysArgXaa                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /product="OTHER"                                       /note= "Amino acid 6 is Xaa wherein Xaa is NH2."                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CysArgValThrCysXaa                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CysSerThrSerCys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CysSerThrSerCysCysSerValThrCysGly                                             1510                                                                          __________________________________________________________________________

What is claimed is:
 1. A polypeptide selected from the group consistingof:

    ______________________________________                                        CSVTCG                                                                        CSVTCG-NH.sub.2                                                               CSVTCGF'      disulfide linked,                                               CSTSCG                                                                        CSTSCG-NH.sub.2                                                               CSTSCG        disulfide linked,                                               CSTSCG-NH.sub.2                                                                             wherein the Cys residues are blocked,                           CRVTCG                                                                        CRVTCG        disulfide linked,                                               CRVTCG-NH.sub.2                                                               RCRVTCG       disulfide linked,                                               CSVTCK                                                                        CSVTCR-NH.sub.2                                                               CSRTCG                                                                        CRVTCG-NH.sub.2                                                                             disulfide linked,                                               CRTSCG-NH.sub.2                                                               CSTSCR-NH.sub.2                                                               CRVTC-NH.sub.2                                                                              and                                                             CSTSCCSVTCG.                                                                  ______________________________________                                    